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International audience; In order to mimic the first steps of lignification, pectin/synthetic lignin (dehydrogenation polymers (DHPs)) interactions were investigated using reconstructing approaches. In artificially lignified bacterial cellulose(BC)/pectin composites, DHP nodules display a more dispersed distribution than in BC mats. Occurrence of covalent linkages between pectin and DHP was investigated by solid state NMR. Non-covalent bonds seem to govern the associative behaviour and stability of the complexes. Studied by small angle neutron scattering, a compaction process of DHP/pectin clusters occurs during polymerization and phase separation of the two polymers was shown at the nanometer scale.

International audience; SEC elution curves of spruce milled wood lignin (MWL) and guaiacyl lignin polymer models (G-DHPs) in NN-dimethylformamide (DMF) exhibited a bimodal elution profile. Light scattering measurements indicated that these elution profiles were due to association effects between the molecules. This became apparent from the determination of high molar masses in the range 10(5)-10(8) g/mol. To study this effect, MWL and DHP were fractionated by precipitation in tetrahydrofuran (THF). The THF-insoluble fractions were found to be the fractions corresponding to the apparent high molar mass part of the DMF elution profiles. The THF-soluble fractions proved to be the less-associated fractions, with lower apparent molecular mass. The individual fractions proved to be rather stable in DMF. Accordingly, the bimodal elution profiles of the starting materials were not the result of an equilibrium between associated and molecular dispersed molecules but of different structures exhibiting a specific and stable association pattern. The different fractions were further characterised by SEC in THF after acetylation to determine molar masses in molecular disperse solutions.

Mechanical and chemical properties as well as microfibril angles of wood tissues from different ontogenetic stages are determined for the neotropical lianas Bauhinia guianensis and Condylocarpon guianense. The mechanical properties include the elastic moduli under bending and under dynamic torsion. The chemical analyses cover (i) the content of cellulose, lignin and hemicelluloses fractions, (ii) the monomeric composition of the uncondensed lignin, and (iii) the composition of the hemicelluloses with respect to neutral monosaccharides. By comparing the wood properties of these lianas with the corresponding properties of wood from self-supporting deciduous trees, common characteristics and differences are revealed. Additionally, the changes in the lignin and polysaccharides fractions as well as the variations in microfibril orientation that occur during ontogeny of the two liana species are discussed with regard to their implications for the mechanical properties of wood.

Thermal treatment is one of the widely used methods for feedstuff preservation. The aim of this study was to evaluate possible cell wall alterations during the industrial-scale drying of alfalfa. Alfalfa is mainly dehydrated and pelleted using two drying treatments. First a thermal treatment at 70 °C is applied to the chopped raw material, prior to the main dehydration procedure which involves a temperature of about 600 °C for a restricted time (15 min). The different steps of the dehydration process did not cause significant changes in alfalfa cell wall composition, but the proportion of labile ether-linked lignin structure (β-O-4) increased after the thermal shock. Chemical fractionation of cell wall carbohydrates showed that pectins were less accessible to extraction with ammonium oxalate after alfalfa drying. Moreover, xylanase (endo enzyme) was more effective on cell walls obtained from heated material. These results suggested that heat processing causes some changes in interpolymer bonds (covalent and hydrogen bonds) between pectins and the lignin/hemicellulose matrix, whereby the cell wall network is modified. © 2002 Society of Chemical Industry

In this paper, multivariate chemometric analysis of fluorescence spectra of juvenile and mature eucalyptus wood samples is used in order to investigate the existence of any correlation between fluorescence data and, the physical/chemical properties, as well as the pulping data of eucalyptus wood. The results obtained demonstrated that the wood of juvenile and adult trees could be discriminated by fluorescence spectroscopy. Data analysis also has shown the existence of good statistical correlation between fluorescence data on the one hand, and ash content, sodium hydroxide solubility, and lignin monomeric composition of the wood samples on the other hand. Moreover, fluorescence spectral data were found to be well correlated with the pulping yield and the Kappa number of the kraft pulps issued from the adult and juvenile eucalyptus wood.

Oxidants formed through the interaction between MnO2 and oxalate accumulated in rotten wood have been evaluated as abiotic agents possibly involved in wheat straw ligninolysis. The hemicellulose and cellulose content of straw remained unchanged, and no release of free soluble materials from lignin or polysaccharides could be evidenced. Structural analysis of oxidized lignin in situ has revealed up to 30% and 10% decrease of β-O-4 linked Guaiacyl and Syringyl units, respectively. Lignin phenolic moieties only were directly oxidized by MnO2/oxalate as no structural alteration was observed within extensively permethylated wheat straw. Modifications were also evidenced at the level of the cell wall linked cinnamic acids present in wheat straw. Esterified phenolic acids were more readily oxidized by Mn complexes than ethers analogues, and disappearance of ferulic moieties was always more pronounced than that of p-coumaric acids. The abiotic Mn oxidants generated from MnO2 and oxalate may therefore significantl...

Lignins definition, potential applications, (macro)molecular structures and properties, analytical characterisations and natural versus induced variability are reviewed with reference to both the biological diversity of lignification patterns in ecotypes, mutants and transgenic plants and the levels of organisation of the lignins polymeric networks. A discrimination between a common primary, guaiacyl type metabolism and a more specific secondary, syringyl type is suggested as a base of this variability and properties.

Times Cited: 10 Cathala, B Aguie-Beghin, V Douillard, R Monties, B Symposium H on Biodegradable Polymers and Macromolecules, at the E-MRS 97/ICAM 97 Conference JUN 16-19, 1997 STRASBOURG, FRANCE 10 SI; International audience; The properties of two DHPs (dehydrogenation polymers) models of lignin are investigated at the air/water interface. Limiting area, relaxation behaviour and dilational modulus are discussed in relation to literature data for plant lignins.

4 tables 3 graph.; International audience; In order to test the reproducibility of the synthesis of dehydrogenation polymer models of lignin (DHPs), four samples obtained by the continous and discontinous synthesis (Zutropf method (ZT) and Zulauf method (ZL)) have been investigated by FTIR, thioacidolysis and GPC.

The monomeric composition of in situ lignins in sorghum, maize and wheat straw stems is investigated by two chemical degradation methods: thioacidolysis and alkaline nitrobenzene oxidation. It is shown that the structural information obtained in the case of lignin characterisation by alkaline nitrobenzene oxidation is strongly dependent on the reaction conditions, namely the temperature and the reaction time, as indicated by the significant variations in the yields of aromatic aldehydes that correspond to the three main constitutive lignin units, p -hydroxyphenyl (H), guaiacyl (G) and syringyl (S). Moreover, the complementary character of the two methods that stems from their different specificities is discussed in view of the molecular heterogeneity of lignins. Finally, the results obtained indicate a significant structural variability of the lignins of the three crops examined.

In plant cell walls, lignins, the second most abundant biopolymers, form 3D crosslinked molecular structures which interact with polysaccharides to form lignin-carbohydrate complexes. The incorporation of these phenolic polymers into synthetic polysaccharide-based composites is currently under investigation. The properties of the final material (mechanical strength, degradability,…) strongly depend on the degree of compatibility between such hydrophilic and hydrophobic molecules. This phenomenon is expected to be in part related to the surface properties of lignins. Organosolve dioxane lignins were isolated from wheat straw and characterized with respect to their HPSEC elution profile ( M n =3.6×10 3 ), total sugar and esterified phenolic acid content (3 and 3.5%, respectively) and to the degradation yield of thioacidolysis (750 μ moles g −1 ). These lignins were then dissolved in a dioxane–water mixture and used for Langmuir trough experiments at the air–water interface. Compression isotherms (pressure/concentration) were obtained at 20°C by compressing the surface deposited layers at specified rates. A particular feature of the interfacial layers of lignin is that surface pressure relaxes strongly when compression is stopped. Thus, the compression isotherms exhibit a surface pressure which is roughly three times higher than the value reached after 48 h relaxation. These data suggest that previously reported isotherms have been determined in conditions far from equilibrium. Dilational moduli, measured at several surface concentrations, range from 13.5 to 72 mN m −1 . These special surface properties should determine a part of the behavior of lignins in blends.

Absorbance at 280 nm was used to determine lignin concentrations in solutions of cell walls from winter wheat whole-crop silage, stems, and straw. Three different cell wall preparations (hot water−organic solvent fiber, ethanol−benzene residue, neutral detergent fiber) were prepared and dissolved in either HCl−triethylene glycol or acetyl bromide. Three phenolic standard solutions were used to calculate lignin concentrations in the wheat materials from absorbance readings: mixtures of phenolic monomers based on aldehydes (HIGUCHI), aldehydes and acids (REEVES), and washed Indulin (WIND). Gravimetric Klason lignin determination on the cell wall preparations consistently gave higher values than spectrophotometric lignin analysis. Material recovered as cell wall was slightly greater for hot water−organic solvent fiber than for neutral detergent fiber and was lowest for ethanol−benzene residue, probably due to partial dissolution of cell wall components with the latter procedure. Acetyl bromide shifted UV sp...

Internodes of maize (Zea mays L, Co125), harvested 5 days after anthesis, were sectioned into five equal parts and samples of sclerenchyma and parenchyma cells mechanically isolated from each section. Phenolic acids and syringyl and guaiacyl degradation products of lignin were released from the walls of the two cell types by microwave digestion with 4 M NaOH. Aryl ether bonded units were selectively released by thioacidolysis. Total phenolic content of cell walls from the youngest (basal) sections were approximately two-thirds of those of the oldest, topmost sections (parenchyma 70·8–99·0 and sclerenchyma 72·5–114·1 mg g-1) indicating that the process of lignification was already well advanced amongst most of the cell walls of the youngest section. The total phenolic content was marginally, but significantly, greater (P

The chemical structure of lignins in Buxus sempervirens wood samples showing a progressive grading from normal wood to reaction wood was investigated by thioacidolysis. The mechanical states of the various samples were assessed quantitatively by measuring the growth strains at the stem surface. Sample woods with the more pronounced compression character showed the highest lignin contents. Buxus lignins are composed of guaiacyl and syringyl units, similar to more evolved angiosperm lignins. The proportion of syringyl units, together with that of labile inter-unit ether bonds, was, however, lower in boxwood lignins than in poplar or birch lignins. More importantly, lignins in boxwood samples with a pronounced compressed character were found to have structural similarities with gymnosperm compression wood lignins, i.e. a higher content of carbon-carbon inter-unit bonds and of p-hydroxyphenyl units, compared with normal wood lignins.

The effect of temperature and time on the recovery yield of the products of alkaline nitrobenzene oxidation of in situ lignins in wheat straw inter-nodes and corresponding leaves were studied. The results showed that significant variations in the yields of aromatic aldehydes (p-hydroxybenzaldehyde, vanillin and syringaldehyde) as well as hydroxycinnamic acids (p-coumaric and ferulic acids) arise from changes in the temperature and the reaction time. The conditions for maximum yield depend on the aldehyde concerned. For example, the yield of p-hydroxybenzaldehyde increases with increasing temperature and reaction time. This phenomenon could be attributed to the low relative reactivity related to the specific electronic structure of the non-methoxylated units and/or to the existence of specific condensation reactions involving the p-hydroxyphenyl aromatic ring. Finally, the results indicate a much higher complexity of Gramineae lignins compared to those derived from hardwoods and softwoods.

The catalytic activity of a new oxidation-resistant Fe(III) meso-tetrakis(pentafluorophenyl)-β-tetrasulfonatoporphyrin [Fe(TF5PS4P)] on spruce milled wood lignin (MWL) and lignin in extractive free...

Summary Lignin is a polymeric constituent of the cell wall that needs to be removed during the paper making process. Bi-specific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) catalyses the O-methylation of caffeic acid and 5-hydroxyferulic acid to ferulic acid and sinapic acid, respectively. These compounds are intermediates in the biosynthesis of the lignin precursors. Therefore, COMTs are potential target enzymes for reducing the amount, or modifying the composition, of lignin in plants. Different antisense and sense constructs have been expressed of a gene encoding a COMT from poplar (Populus trichocarpa x P. deltoides) in a P. tremula x P. alba clone under the control of the cauliflower mosaic virus 35S promoter. From all analysed transformants, four lines transformed with an antisense construct had a reduced COMT activity. Two showed a 50% reduction of COMT activity, which altered only slightly the monomeric composition. In the two other transformants, the COMT activity was reduced by 95%. In the latter case, the syringyl/ guaiacyl ratio (S/G) was reduced by sixfold (due to a decrease of S and an increase of G), as analysed by thioacidolysis. A new component of lignin, the 5-hydroxyguaiacyl residue, was detected among the thioacidolysis products. Moreover, in contrast to the white/yellow colour of wild-type wood, the xylem of the transgenic lines with a 95% reduction of COMT activity was pale rose. A similar phenotype was observed in brown-midrib mutants of maize and sorghum, known for their altered lignification. Although the lignin composition was consistently modified, the lignin content of the transgenic poplars was similar to that of the controls.

The molecular structure of lignins (occurrence of condensed and non-condensed monomeric units and associations with phenolic acids by ether and ester linkages) have been reviewed in this paper. Critical aspects of chemical determination of lignin content and monomeric composition have been discussed in the case of nitrobenzene oxidation and thioacidolysis. As an example of assessment of lignin biodegradation by peroxidases, lignin peroxidase and horseradish peroxidase have been compared, indicating that horseradish peroxidase is a ligninolytic enzyme, confirming the influence of the physical state of lignins on their biodegradability and the general difficulty to characterize biomacromolecules.

Organosolv lignin fractions were used as substrate for further investigations on the effect of bleaching and photoyellowing on lignin. These lignins were extracted from spruce TMP, bleached TMP (BTMP) and bleached and yellowed TMP (YBTMP). Furthermore, lignin fractions from TMP were also directly submitted to bleaching conditions and lignin fractions from BTMP to irradiation in the solid state. These lignin samples were all analyzed by thioacidolysis and FT-IR spectroscopy. The bleaching did not substantially change the lignin polymer but considerably destroyed some chromophoric structures such as coniferaldehyde end-groups. On the other hand, the photoyellowing resulted in marked degradations of lignin, shown by a decrease in the amount of β-O-4 and β-1 interunit bonds and a substantial increase in vanillin and vanillic acid end-groups and in catechol units.

Formation of standard guaiacyl-syringyl lignin has been shown, by thioacidolysis, during healing and suberization of wound potato tuber discs. Recovery of p -hydroxybenzaldehyde after nitrobenzene oxidation has been related to the presence of tyramine. Variations in the relative proportion of hydroxyphenyl, guaiacyl and syringyl degradation products obtained after nitrobenzene oxidation, thioacidolysis and alkaline hydrolysis suggest the formation, during suberization, of a complex phenolic network linked to the cell wall.

This paper reports the structural investigation of a series of in situ spruce lignins in wood, thermomechanical pulp (TMP) and the corresponding bleached (BTMP) and photoyellowed (YBTMP) samples. This was achieved by using an original two-step degradative technique, thioacidolysis followed by Raney nickel desulfuration. The determination of thioacidolysis monomeric and dimeric products allowed an estimate of various lignin building units and interunit bonds, respectively. It was observed that the thermomechanical and bleaching treatments did not affect spruce lignin structure to an appreciable extent. On the contrary, the photoyellowing treatment caused marked structural changes in lignin, particularly when run in severe conditions. These changes were essentially a decrease in the amount of β-O-4 and β-1 interunit bonds and a relative increase in catechol units and in vanillin end-groups.

Various spruce mechanical pulps were subjected to delignification with ethanol-water (1 : 1, v / v ) containing 0.1 M acetic acid at 175°C in a flow- through reactor. A thermomechanical pulp (TMP) and the corresponding samples derived from its bleaching (BTMP) and yellowing ( YBTMP ) treatments were delignified to a similar extent, about 70% of delignification degree, as compared with spruce wood. A series of five successive lignin fractions was recovered from each pulp sample and then characterized by various analytical methods. Large structural variations were observed within these fractions. The number of phenolic structures appeared to be an important factor influencing the dissolution of lignins in the ethanol-water medium. These lignin fractions were found to be different in the amount of β-aryl ether structures and in the relative importance of main carbon-carbon bonding patterns. The results are discussed in relation to the lignin fractionation in the flow-through reactor.

The GC-MS identification of the main dimers recovoered after thioacidolysis, then desulphuration over Raney nickel of spruce lignin allowed to characterize the various types of condensed linkages in softwood lignin. The major carbon-carbon bonds were observed to-be represented by the β-5, 5-5 and β-1 interunitary linkages established between two guaiacyl units, in agreement with literature data. Besides, minor linkage patterns were represented by dimers provided with the diphenyl ether, the phenylisochroman and the tetrahydrofuran structures

The rate of oxidation of spruce milled wood lignin by Phanerochaete chrysosporium was investigated with precipitated lignin as well as lignin finely dispersed in water (colloidal lignin). H2O2 consumption rates in the course of lignin oxidation in the presence of purified lignin peroxidase were much higher with colloidal lignin compared with precipitated lignin. Characterization of reaction products and molecular-size distribution confirms that reaction rate of lignin peroxidase with 14C-lignin is strongly dependent on the physical state of lignin. The lignin peroxidase catalyse mainly repolymerization of colloidal lignin. However, in the presence of veratryl alcohol, both polymerization and depolymerization were observed.

Heartwood of Quercus robur L. contains about 10% (w/w), of hexahydroxydiphenoyl esters (ellagitannins). Five of the most abundant hexahydroxydiphenoyl esters were purified by a combination of chromatography on Sephadex LH-20 and high-performance reversed-phase liquid chromatography. Two of them were identified as castalagin and vescalagin.

International audience; Microscopic and chemical changes of hemp bast fibers were studied during the maturation from vegetative to grain maturity stages at both apical and basal regions of the stems. The content of protein was the main factor related to fiber maturation, whereas increased proportions of mannose and glucose and decreasing levels of galactose were also highly significant. Enhanced glucose deposition in apical fibers could be related to the gradual thickening of the fibers, whereas in basal regions the thickness of the fibers nearly reached the maximum at vegetative stages. In contrast, the extent of lignification remained close to 3-4% during plant growth. Hemp fiber lignins were rich in guaiacyl units and would be rather condensed in nature. In addition, the proportion of p-hydroxyphenyl units displayed a constant decline during maturation. A progressive chemical fractionation of hemp fibers provided further insights to the occurrence and nature of noncellulosic polysaccharides. Notably, these data pointed out that maturation is accompanied by a significant increase in water- and alkali-soluble components containing glucose- and mannose-related polymers and a decrease in arabinose and galactose components disrupted by diluted hydrochloric acid. Taken together, chemical features of the noncellulosic components suggest that the architecture of hemp fibers differs slightly from that of the more widely studied flax fibers.

International audience; Kraft lignin and vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol, used as model compound) were treated under acidic conditions (sulphuric and hydrochloric acid). Hydroxyl content and molar masses of dichloromethane extracted fractions were determined. The acidic treatment decreased the aliphatic hydroxyl content and so lowered the polarity of the lignin molecules without derivatization. Antioxidant properties in polypropylene of the treated fractions were increased compared to that of untreated ones.

Lignocellulosic fractions from wheat straw were used as natural fillers in composites of a polyolefin (a copolymer of polyethylene and polypropylene) and a biodegradable polyester [poly(butylene adipate-co-terephthalate)]. The mechanical properties of these injected composites were investigated with tensile and impact testing. A reinforcing effect of wheat-straw residues was found for both types of composites. Compared with the polyester-based composites, the polyolefin composites were more brittle. The addition of compatibilizing agents (γ-methacryloxypropyltrimethoxysilane, maleic anhydride modified polypropylene, and stearic acid) did not improve the properties of the polyolefin composites. The surface properties were studied with contact-angle measurements, and poor interfacial adhesion was found between the hydrophilic lignocellulosic filler and the hydrophobic polyolefin matrix. Thermal characterization revealed the formation of low intermolecular bonds between the polyester matrix and the lignocellulosic filler, in agreement with the surface tensions results and scanning electron microscopy observations. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 428–436, 2004

International audience; The effects of heat treatments used to dry alfalfa stems were investigated. Heating at 70 or 100 °C caused no major change in the cell wall composition, but xylanase had lower activity on the cell wall of heated material and the amount of xylose released varied with the temperature used. Chemical fractionation of cell wall carbohydrates showed that the main changes occurring during stem dehydration concerned pectic polymers and probably hemicelluloses. There was less material soluble in ammonium oxalate from alfalfa heated at 100 °C than from fresh alfalfa. The results suggest that heat processing causes some changes in the cell wall network. Environmental scanning electron microscopy was used to examine fully hydrated tissues at high resolution. There was cell distortion without disruption of cell walls as water was lost.

The Arabidopsis thaliana sam1 gene encoding S-adenosylmethionine synthetase (EC 2.5.1.6) was transferred to flax (Linum usitatissimum) cells via Agrobacterium tumefaciens. This enzyme catalyses the conversion of methionine to S-adenosylmethionine (SAM), the major methyl group donor in living cells. The aim of this work was to study the consequences of an increased SAM-synthetase (SAM-S) activity in transgenic cell lines on both the production of mono- and dimethoxylated lignin monomers and the degree of methylesterification of pectins. Hypocotyls were cocultivated with Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the pO35SSAM binary vector carrying the sam1 gene under the control of the 35S promoter and the nptII gene for selection of putative transformed cells. Most of the transgenic cell lines exhibited a significant (up to 3.2-fold) increase in SAM-S activity compared to the controls. The results showed that for the cell lines analysed this transformation had no effect on caffeic acid O-methyltransferase (COMT, EC 2.1.1.68) in vitro activity, degree of methoxylation of lignin precursors or lignin deposition, pectin methyltransferase (PMT, EC 2.1.1) in vitro activity, but led to an increase of pectin methylesterification in friable and fast-growing transgenic cell lines.

Introduction Historical Outline Occurrence in the Plant Kingdom According to Type of Plant According to Plant Tissue Contribution to Dry Matter Annual Global Synthesis Ultrastructure of Wood and Lignocellulose Role of Lignin in the Biopolymer Matrix Interactions with Other Cell-wall Polymers Functions of Lignin Stability of Plant Tissues Protection against Biodegradation Regulation of Water Transport Other Functions Related to Plant Evolution and Systematics Precursors of Biosynthesis Biosynthesis Transport Established Pathways Radical Polymerization Labeling Experiments Recent Findings, Hypotheses, and Controversies Regulation of Lignification Expression of Enzymes and Genes Effects of Genetic Manipulation Patents Keywords: algae; angiosperm; biodegradation; biomechanic; plant cell wall; cellulose; cryptogram; plant evolution; fern; grass; gymnosperm; hemicellulose; humification; lignins; lignin–polysaccharide complex; monocotyledon; moss; phenolic acids; plant systematic; pectin; structural protein; suberin; water transport; wood

International audience; Coniferyl alcohol was polymerised in the presence of pectin and a pectin-DHP complex was isolated. Characterisation of the complex has demonstrated that DHP (20% wt) was covalently linked by ester bonds to the pectin. The water sorption behaviour of the pectin-DHP complex was determined at several relative vapour pressures and compared with those obtained on pectin, DHP and a blend of both polymers in the same proportion as in the complex. The complex exhibited a lower hydrophilicity resulting from three associated phenomena: grafting, pectin-DHP interactions and the formation of a chemical network.

International audience; Dehydrogenation polymers (DHPs. lignin model compounds) were synthesized in the presence of increasing pectin concentrations using two different methods. The first method ('Zutropfverfahren'. ZT) consists in the slow adding of monomers whereas in the second method ('Zulaufverfahren', ZL) all the reactants are added simultaneously. DHPs solubility increases with the pectin concentration in the ZT experiments and remains stable in the ZL experiments. Covalent bonds between pectin and DHP are formed during ZT polymerization resulting in lignin carbohydrate complex (LCC) which keeps the unbound DHPs in solution by the formation of aggregate or micelle-like structures. In contrast LCC are not formed during the ZL process which behave like the DHP reference. The ZT DHP molar masses increase observed is attributed to the reactivity of the high molar mass polymer solubilized by the: LCC whereas ZL higher molar mass polymers are precipitated out of the solution and cannot react further.

Brown-midrib (bm) mutants of maize have modified lignin of reddish-brown colour. Although four independent bm loci are known, only one of the mutant genes has been previously identified. We report here that maize bm1, one of the less characterised mutants, shows severely reduced CAD activity in lignified tissues, resulting in the production of a modified lignin. Both the total lignin content and the structure of the polymer are altered by the mutation. We further describe the isolation and characterisation of the maize CAD cDNA and mapping of the CAD gene. CAD maps very closely to the known location of bm1 and co-segregates with the bm1 locus in two independent recombinant inbred populations. These data strongly support the premise that maize bm1 directly affects expression of the CAD gene.

In addition to morphological factors (height of the plant, proportion of stems), alfalfa quality is related to several biochemical factors, especially the content and monomeric composition of the cell wall components. This work was aimed at studying internode development in relation to the chemical composition and fractionation of cell walls. Analyses were performed on the fourth apical internodes (elongating), the two following ones (onset of cambial activity), and bottom internodes (mature). Deposition of guaiacyl type lignin occurred in the pectin rich cell walls of apical internodes. The onset of cambial activity corresponded to cell wall accumulation of syringyl-guaiacyl lignin, xylans and/or glucuronoxylans, and cellulose. Such events were related to the thickening of secondary cell walls which proportion increased in mature internodes. Chemical fractionation of the cell walls allowed the release of a high content of water soluble pectins from apical internodes, whereas alkalis were more efficient in extracting heteroxylans from bottom internodes. A partial release of lignin from the cell walls also occurred during the extraction steps. Lignin solubilization was enhanced by the cleavage of labile-ether lignin structures and appeared closely related to the extent of internode differentiation along the stem. Data gained from chemical fractionations evidenced that lignin-xylan-pectic complexes accumulated progressively in secondary wall rich tissues.

Peroxidases and laccases are key enzymes in the lignin biodegradation process. They oxidize phenolic and non-phenolic lignin model compounds into their phenoxy and cation radicals, respectively. Further non-enzymatic evolution lead then to various C-C and ether bond cleavages. Nevertheless, almost no information on the structural alterations undergone in vitro or in situ by lignin after enzymatic catalysis is available. We report here on the molecular structure of lignin oxidized by various (per)oxidasic systems. The oxidizability of phenolic and non-phenolic structures of the guaiacyl and syringyl type in the lignin network will be discussed as well as the modification of the macromolecular properties of the polymer oxidized in situ or in isolated state.

In the present study, we have performed chemical investigations of the stem cell walls during internode maturation in order to study the growth dynamics of alfalfa and the deposition of the main cell wall components (polysaccharides and lignins). Internode cell walls were analysed by chemical fractionation using a mild delignification step aiming at sequential removal of polysaccharides and lignins. Delignification facilitated the subsequent removal of the xylose-rich polysaccharides by NaOH extraction as previously shown. This trend was more pronounced in the case of older internodes which have a larger proportion of secondary tissues containing syringyl type lignins in contrast to younger ones which are mainly composed of primary tissues containing guaiacyl type lignins and pectin rich cell walls.

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in the biosynthesis of the lignin precursors, the monolignols. We have down-regulated CAD in transgenic poplar (Populus tremula X Populus alba) by both antisense and co-suppression strategies. Several antisense and sense CAD transgenic poplars had an approximately 70% reduced CAD activity that was associated with a red coloration of the xylem tissue. Neither the lignin amount nor the lignin monomeric composition (syringyl/guaiacyl) were significantly modified. However, phloroglucinol-HCl staining was different in the down-regulated CAD plants, suggesting changes in the number of aldehyde units in the lignin. Furthermore, the reactivity of the cell wall toward alkali treatment was altered: a lower amount of lignin was found in the insoluble, saponified residue and more lignin could be precipitated from the soluble alkali fraction. Moreover, large amounts of phenolic compounds, vanillin and especially syringaldehyde, were detected in the soluble alkali fraction of the CAD down-regulated poplars. Alkaline pulping experiments on 3-month-old trees showed a reduction of the kappa number without affecting the degree of cellulose degradation. These results indicate that reducing the CAD activity in trees might be a valuable strategy to optimize certain processes of the wood industry, especially those of the pulp and paper industry.

Stem bark and pith of sweet sorghum were analysed with reference to their sucrose, simple reducing sugars, cellulose, hemicelluloses, lignin and associated phenolic acids contents. Moreover, lignin monomeric units (guaiacyl and syringyl) engaged in non-condensed structures were characterized by thioacidolysis, whereas cell wall associated phenolic acids (p-coumaric and ferulic acids) were estimated by alkaline hydrolysis at 170°C. The results obtained showed that bark and pith are heterogeneous as far as their chemical composition and the structure of their chemical components are concerned. In particular, the pith content in water soluble sugars is twice as high compared to the one in the bark, whereas bark is enriched in lignocellulosic fibres. Bark lignin is twice more important in content and less condensed in structure compared to pith lignin. P-coumaric acid is the predominant p-hydroxycinnamic acid associated to the cell walls, whereas ferulic acid is present in significant quantities.

The removal of lignin in paper manufacture is a toxic and energy-requiring process. For the paper industry and for the environment, it would be very beneficial to process trees with less lignin or with lignin that is more easily ex tractable. To achieve this goal, we are down-regulating the activity of a number of lignin biosynthesis enzymes in poplar by genetic engineering and studying their role in determining the quantity and quality of lignin. Here, our recent results obtained in this field are summarized with emphasis on bispecific caffeic acid/5-OH-ferulic acid-O-methyltransferase, cinnamoyl-CoA-reductase, caffeoyl-CoA-O-methyltransferase, and peroxidase.

Cinnamyl alcohol dehydrogenase (CAD) catalyses the last step in the biosynthesis of the lignin precursors, which is the conversion of cinnamaldehydes into cinnamyl alcohols. A poplar (P. trichocarpa x P. deltoides)CAD cDNA was introduced in sense and in antisense orientation into P. tremula x P. alba via an Agrobacterium- mediated transformation. The CAD activity was reduced by 70% in the xylem tissue of three antisense and two co-suppressed lines. A red coloration of the xylem was detected in all poplar lines with a residual CAD activity of less than 60 %. The lignin content and the lignin composition (syringyl/guaiacyl) were similar in co-suppressed, antisense CAD and control lines. However, chemical properties of the cell wall were different. First, a phloroglucinol staining showed a different coloration in the xylem of the down-regulated CAD poplars, being red-brown as compared to the typical pink staining of the control xylem. Second, after NaOH treatment, lignin could be precipitated by acidification of the alkali-soluble fraction of the cell wall of the red poplars but not of that of the white poplars. A spectral characterisation of the alkali fraction from red poplars showed an increased level of vanillin and syringaldehyde. Third, pulping experiments showed less residual lignin in the pulp of CAD down-regulated poplars.

The effects of the maize brown-midrib genes bm3 and bm1 on feeding value and agronomic traits were investigated by animal studies and biochemical approaches. When sheep were fed bm3 silage, an increase in digestibility was observed, equal to 2.4 points for digestibility of organic matter (DOM), and 8.1 points for digestibility of crude fiber (DCF). Phenotypic variation was reduced for all observed traits of bm3 hybrids. This allowed the assumption that the bm3 gene acted by levelling to a similar value of almost 74% DOM, quite independently of the original value of the normal counterpart. The bm1 hybrids were intermediate between normal and bm3 ones for DOM, DCF and intake, but the dry matter yield was not reduced for bm1 hybrids. From experiments with young bulls, feed efficiency of INRA260 bm1 was not significantly improved, compared with normal INRA260, but INRA260 bm3 was a lot more efficient. Despite its lower adverse effect on agronomic characteristics of hybrids, the bm1 allele did not appear to be of any interest in improving the silage maize feed efficiency. Variations for lignin content due to the bm3 gene was similar for the 3 hybrids studied, despite their variation for susceptibility to root and stalk lodging. Ratios and content of alkali-labile hydroxycinnamic acids behaved similarly for the 3 hybrids, with a low yield of para-coumaric acid. Structural studies of lignin, after thioacidolysis, strengthened the results previously obtained, with a clear decrease of syringyl units, and the occurrence of the 5-hydroxyguaiacyl residue. The lignin of bm3 genotypes also appeared as a more condensed polymer than that of the normal types. The decrease of lignin content was observed in only 1 bm1 hybrid and appeared the same between normal and bm1 hybrids as between normal and bm3 hybrids. The lower yield of para-coumaric acid, and the equal yield of ferulic acid after alkaline hydrolysis of bm1 material were in good agreement with previous work. After thioacidolysis, the release of monomeric syringyl units was similar between bm1 and bm3, about 30% of the normal counterpart. However, it was very different for guaiacyl units, with a large decrease of the release of these units from the bm1 hybrid. The ratio between syringyl and guaiacyl units was about 0.3 for bm3 hybrids and about 1.3 for the bm1 hybrid. The lignin of this bm1 hybrid appeared as a very condensed polymer compared with the normal type. A large variation for digestibility was also found among normal hybrids. However, inbred lines inducing high digestibility often included unfavorable agronomic traits, such as poor yield, susceptibility to lodging or stalk rot. Second or third cycle lines following pedigree breeding might combine all traits, even if stalk standability and stalk digestibility are partly antagonistic. This method may be considered as easier than the bm3 method, but at present, the 2 ways are probably complementary, and worth investigating for improving silage digestibility, and simultaneously increasing knowledge on silage maize digestibility., Les effets des gènes brown-midrib bm1 et bm3 sur la valeur alimentaire et les caractéristiques agronomiques d’hybrides de maïs ont été étudiés à partir d’essais sur animaux et de mesures de composition biochimique de la lignine. Sur moutons, les hybrides bm3 conduisent à une amélioration moyenne de la digestibilité de la matière organique (DOM) voisine de 2,4 et de celle de la cellulose brute (DCF) voisine de 8,1 points. La variabilité phénotypique est nettement réduite entre hybrides bm3, comparée à celle observée pour les quasi isogéniques normaux, pour la quasi-totalité des caractères étudiés. En particulier, il semble que la présence du gène bm3 conduise à augmenter la DOM des hybrides à une valeur proche de 74%, quel que soit le niveau de digestibilité de l’hybride normal de départ. Les hybrides bm1 se situent de façon intermédiaire entre les formes normales et bm3, sauf pour la productivité en biomasse qui est équivalente à celle des normaux. Mais des essais de croissance comparée de taurillons nourris avec des ensilages de maïs INRA260, INRA260 bm1 et INRA260 bm3 montrent que la forme bm1 ne permet pas, contrairement à la forme bm3, une amélioration notable des performances animales. La réduction de teneur en lignine a été semblable pour les 3 hybrides bm3 étudiés, alors que ces hybrides présentaient des différences importantes de sensibilité à la verse ou à la casse. Les teneurs et rapports entre acides hydroxycinnamiques libérés après hydrolyse alcaline étaient semblables chez les 3 hybrides, avec une libération faible d’acide para-coumarique caractéristique des génotypes bm3. La thioacydolyse confirme que la lignine des plantes bm3 est plus pauvre en unités syringyl, avec, de plus, la présence d’une unité 5-hydroxyguaiacyl. La lignine des plantes bm3 est un polymère plus condensé que la lignine des plantes normales. Observée à partir d’un seul hybride bm1, la diminution de la teneur en lignine a été semblable chez les plantes bm1 et bm3. Après thioacidolyse, la libération d’unités syringyl était équivalente entre bm1 et bm3. Mais la libération d’unité guaiacyl était beaucoup plus faible pour la lignine des plantes bm 1, avec des rapports syringyl/guaiacyl voisins de 0,3 pour les bm3 et de 1,3 pour les bm1. La lignine des plantes bm1 est également un polymère hautement condensé. Une variabilité importante pour la DOM et la DCF existe aussi entre hybrides normaux. Toutefois, les hybrides les plus digestibles présentent en général des défauts agronomiques importants, qu’il sera sans doute possible de corriger au niveau de lignées de second ou troisième cycle. Actuellement, on peut considérer que les voies normales et bm3 sont deux approches complémentaires de l’amélioration et de la compréhension de la digestibilité du maïs ensilage.

Oxidation of spruce milled-wood lignins in colloidal state catalyzed by horseradish peroxidase (HRP) and lignin peroxidase (LiP) in the presence of H 2 O 2 was compared in nonbuffered dimethyl-formamide/water. Lignins were characterized for their hydrodynamic properties by size-exclusion chromatography and structural bonding pattern by analysis after thioacidolysis. In contrast to LiP, HRP did not induce modification of lignin hydrodynamic properties. The lignin content in β-O-4-linked guaiacyl monomers and dimeric structures, however, decreased after oxidation by the two enzymes, indicating large structural changes in the polymer. Preferential degradation by both LiP and HRP of β-5 and β-1 lignin dimeric units was also observed. This suggests a greater susceptibility to enzyme oxidation, as compared to the 5-5′ and 4-O-5 lignin substructures. In conclusion, the action of HRP on the lignin polymer is similar in many respects to that of fungal LiP, but is distinctly different in its inability to cause a net destructuring of the macromolecular three-dimensional network.

Cell wall-linked phenolics were investigated in maize internodes located at three positions of the stem (top-middle-bottom). While the lignin content did not change drastically with position, the amount of cell wall-ester linked p-coumaric acid sharply increased from the top to the bottom internodes of the stem. Conversely, the saponified ferulic acid content remained relatively unchanged along the stem. Moreover, the highest syringyl content of the β-O-4-lignin structures was found in the basal (most mature) internode. Therefore, enhanced p-coumaric esterification of the cell wall and preferential deposition of syringyl units in the lignin polymer might indicate an extended maturity stage of the cell wall of maize internodes. The bm3 mutation in the three maize hybrids is expressed by lignin reduction and ester-bound p-coumaric decrease in the mutant lines. Furthermore, all bm3 hybrids synthesised lignin polymers which were characterised by a very low S/G molar ratio (0.16-0.43). This ratio originates from the substantial reduction of the syringyl unit content in β-O-4-lignin structures compared with the normal lignin. The occurrence in the same range of the 5-hydroxyguaiacyl unit in bm3 lignin from each type of hybrids was noteworthy, demonstrating the high heritability of the bm3 mutation at the molecular level. The alkaline solubility of lignin was greater for the three mutant lines compared to the normal cultivars. Furthermore, the alkali-labile fraction of lignin of both normal and mutant lines had a monomeric composition which was consistent with the non-condensed structures of in-situ lignin.

Cell wall might be considered as a composite material, made of fibre (cellulose), a matrix (lignin, hemicellulose) and fillers (water, simple organics, tannins). Its properties depend both on plant anatomy (heterogeneity and anisotropy of organs, tissues, cells and cell wall layers) and on the structure of the macromolecules, their orientational order, their associations and the resulting supramolecular structures as particularly discussed in the case of lignin networks. From selected examples, it is shown that natural or artificial lignocellulosic compounds such as woods, straws or reconstituted woods (fibre and particle boards) can be described as composite materials. Their most typical physico-chemico-mechanical properties are: shrinkage and water swelling behaviour, collapse in relation to extractive and tannin content, improvement of dimensional and biological stability by chemical modifications, anisotropic properties of orientated strand boards and fibre-reinforced polyester-based composites, and lastly, biological properties (digestibility, extensibility). As for synthetic composite materials, such an approach is a prerequisite for industrial production of competitive lignocellulosic products.

The hypothesis that auxin (IAA) and gibberellic acid (GA(3)) control the formation of lignin is confirmed for the primary phloem fibers and for the secondary xylem in the stem of Coleus blumel Benth. Indoleacetic acid alone, or a combination of high IAA/low GA(3) (w/w), induced short phloem fibers with thick secondary walls, that contained lignin rich in syringyl units (high ratio of syringyl/guaiacyl). On the other hand, a combination of high GA(3)/low IAA (w/w), which promoted the differentiation of long phloem fibers with thin walls, decreased the relative content of the syringyl units (low syringyl/guaiacyl ratio). In the secondary xylem, these hormonal treatments yielded only slight changes in the noncondensed monomeric guaiacyl units, confirming the relative stability of the guaiacyl lignification pattern in this tissue. In the xylem, indoleacetic acid alone, or a combination of high IAA/low GA(3) induced lignin poor in syringyl units (low syringyl/guaiacyl ratio). A combination of high GA(3)/low IAA promoted a relatively slight increase in syringyl yield, indicating greater responsiveness of the syringyl lignification pattern to growth regulators. The possible functional and technological significance of our results is discussed.

L'addition d'eau a une solution de lignine dans le DMF entraine la precipitation d'une partie de la lignine et le passage d'une autre fraction dans un etat colloidal. Toute la lignite presente peut etre transformee dans cet etat par des cycles successifs de solubilisation precpitation de la lignine non-colloidale. Cette lignine colloidale ne presente pas de difference significative de structure moleculaire ni de distribution de masse moleculaire par rapport a la lignine native, mais sa reactivite dans l'oxydation par la peroxydase de la lignine est beaucoup plus elevee